We investigated the microstructural and hardness changes in austenitic stainless steel after Fe ion irradiation at 400, 300, and 200 °C using transmission electron microscopy (TEM) and nanoindentation. The size of the Frank loops increased and the density decreased with increasing irradiation temperature. Radiation-induced segregation (RIS) was detected across high-angle grain boundaries, and the degree of RIS increases with increasing irradiation temperature. Ni–Si clusters were observed using high-resolution TEM in the sample irradiated at 400 °C. The results of this work are compared with the literature data of self-ion and proton irradiation at comparable temperatures and damage levels on stainless steels with a similar material composition with this study. Despite the differences in dose rate, alloy composition and incident ion energy, the irradiation temperature dependence of RIS and the size and density of radiation defects followed the same trends, and were very comparable in magnitude.